The fungal cell wall is a dynamic structure that grows with the cell and imparts shape to it. For that reason, we have used for many years the cell wall of budding yeast as a model for morphogenesis. The wall is essential for cell survival, because in its absence the internal turgor pressure would cause cell lysis. Its resilience is due to the cross-links between its components, polysaccharides and mannoproteins. Thus, the major structural component, beta(1-3)glucan, is linked to beta(1-6)glucan, to which mannoproteins are attached. Chitin, an acetylglucosamine polymer, is joined to both beta(1-3) and beta(1-6)glucan (1). Chitin is a minor component of the cell wall, but it is necessary for yeast survival. Most of the chitin is found in a ring that forms at the neck between mother and daughter cell at budding. Later in the cell cycle, at cytokinesis, a chitin primary septum is formed. Finally, after septation, some chitin appears all around he cell wall of the daughter cell. Synthesis of the chitin in the primary septum is catalyzed by chitin synthase 2, that of all the other chitin by chitin synthase 3 (Chs3). Another ring present at the mother-daughter neck is assembled by five proteins called septins. The septin ring recruits proteins that participate in cytokinesis and is required for cytokinesis. The function of the chitin ring was unknown, because cells defective in Chs3 grow well and show only minor morphological defects. However, about 8 years ago we found that chs3 mutants were synthetically lethal with a septin mutant, cdc11, or with a mutant of the protein kinase Cla4, in whose absence the septin ring is partially defective (2). In an attempt to understand the synthetic lethality, we allowed single cells of a cla4delta strain or the cdc11 mutant to bud either in normal medium or in the presence of Nikkomycin Z, a specific inhibitor of Chs3. The control cells budded normally, but those in the presence of the inhibitor gave rise to elongated buds with very wide necks. Those buds never completed cytokinesis. A recent experiment showed the same final result if Nikkomycin was washed out just after bud emergence, suggesting that all subsequent morphological defects were caused by elimination of the chitin ring. Since the effect of the inhibitor is only seen with septin impaired cells, we concluded that the septin and the chitin ring have a redundant function in preventing neck widening, i.e. cell wall growth at the neck. The septins, which have been shown to act as barriers to the movement of some proteins between mother and daughter cell, may act by barring access to the neck of proteins needed for growth. The chitin ring might function either in a mechanical way, by impeding expansion of the cell wall, or in a chemical way, by capping the nonreducing ends of beta(1-3)glucan, thus preventing further beta(1-3)glucan metabolism and the attachment to those same ends of beta(1-6)glucan and mannoproteins. In this way, growth of the cell wall would be blocked. We preferred the chemical explanation, because chs3delta mutants do not show a widened neck, despite the turgor pressure. This hypothesis makes two predictions;one is that at the neck most of the chitin should be bound to beta(1-3)glucan, whereas it may be mainly attached to beta(1-6)glucan in lateral walls. We were indeed able to confirm this prediction (3). The other consequence of the hypothesis is that it is the binding of chitin to beta(1-3)glucan and not the mere presence of the chitin ring, that controls growth at the neck. Work in collaboration with the laboratories of Javier Arroyo and of Vladimir Farkas showed first that the two proteins Crh1 and Crh2 were redundantly required for attachment of chitin chains to beta(1-6)glucan (4). These proteins act as transglycosylases, transferring chitin chains to glucan (5). Later, I was able to show that Crh1 and Crh2 also catalyze the binding of chitin to beta(1-3)glucan (6). These findings enabled us to test the second part of our hypothesis, according to which cells lacking bonds between chitin and glucan should be as defective as those lacking the chitin ring altogether. Note that , despite the synthetic lethality between chs3 and cla4 mutants, we had been able to isolate a double mutant chs3delta cla4delta, which probably survived thanks to the presence of some suppressor. This mutant grew very slowly and showed and extremely aberrant morphology, with wide necks, very elongated buds and frequently bloated cells. We deleted, in a wild type strain of the same genetic background, CLA4, CRH1 and CRH2. The resulting triple mutant showed a morphology similar to that of the cla4 chs3 strain, despite a chitin content 4 times that of wild type and the presence of chitin rings. Measurement of neck diameters showed a large increase relative to wild type in both mutants. Transformation of the triple mutant with wild type CRH genes restored normal morphology, but transformation with CRH genes carrying mutations in the putative active site did not. We also found that inhibition of Crh2 with chitin oligosaccharides in vivo, in a cla4delta crh1delta strain led to widened necks and elongated buds. It seemed probable that bud elongation in the triple mutant was a consequence of neck widening, which triggered the so-called morphogenetic checkpoint, stopping the cell cycle before the transition between polar and isometric growth (7). The protein kinase Swe1 has a major role in this checkpoint. Deletion of SWE1 in the triple mutant largely abolished bud elongation but not neck widening, showing that the two processes can be separated. In conclusion, we have uncovered a case in which the chemical bond between two substances, chitin and glucan, has a profound effect on the control of morphogenesis. This conclusion, which may apply to other systems, is consistent with our hypothesis about the effect of the chitin ring on neck growth, but would still be valid with other explanations of ring function. (1) Cabib, E., Roh, D.-H., Schmidt, M., Crotti, L.B., and Varma, A. (2001) J. Biol. Chem. 276, 19679-19682. (2) Schmidt, M., Varma, A., Drgon, T., Bowers, B., and Cabib, E. (2003) Mol. Biol. Cell 14, 2128-2141. (3) Cabib, E., and Durn, A. (2005) J. Biol. Chem. 280, 9170-9179. (4) Cabib, E., Blanco, N., Grau, C., Rodrguez-Pea, J.M., and Arroyo, J. (2007) Mol. Microbiol. 63, 921-935. (5) Cabib, E., Farkas, V., Kosk, O., Blanco, N., Arroyo, J., and McPhie, P. (2008) J. Biol. Chem. 283, 29859-29872. (6) Cabib, E. (2009) Eukaryot. Cell 8, 1626-1636. (7) Lew, D.J. (2003) Curr. Opin. Cell Biol. 15, 648-653.